A sensitive method for light microscopic localization of brain receptors by in vitro autoradiography was developed previously in this laboratory. By this method we have mapped the locations of drug and neurotransmitter receptors in the brains of rats and other vertebrates, including primates. Radiolabeled ligand binding using subtype-selective assay conditions confirmed the existence of anatomically distinct mu, delta, and kappa opiate receptor subtypes. A similar strategy led to the visualization of separate tachykinin receptors marked by radiolabeled substance P and eledoisin. Immunohistochemistry is used to compare the distributions of putative neurotransmitters and their receptors. The relationship of these localization patterns with other markers of brain heterogeneity, provided by tract tracing and enzyme staining, allows hypotheses about functional circuitry in the central nervous system. Physiological activation of neurochemically defined systems may lead to receptor occupation or regulation, which can be revealed and localized by in vivo as well as in vitro autoradiographic techniques.